Studies completed in this project indicate that cytoplasmic membranes prepared from sensitive bacteria treated with colicin K or El but not E3 are impaired in their ability to catalyze ATP-linked transhydrogenase reaction and are missing or contain reduced amounts of certain classes of the membrane proteins. The activities of several electron transport enzymes and ATPase enzyme were tested and found not to be altered in the membranes of the colicin treated cells. These results indicate that colicins K and E1 interfere with the coupling of ATP-hydrolysis to the generation of an energized membrane state that is required to drive the reaction or with the utilization of the energized state. One or more of the membrane proteins, affected in colicin treated cells, might participate in energy transduction processes by acting as coupling factors. To test this possibility the membrane proteins will be purified and their ability to reconstitute the activity to impaired membranes will be tested. The results should lead to the identification of the primary target of colicin K and E1 in the bacterial cytoplasmic membranes. The addition of purified colicin K or E1 to cytoplasmic membranes in vitro did not result in an impairment of the membrane ATP-linked activity. Either the colicin cannot interact directly with its targets in the membrane, or the targets are masked. To test these possibilities, the colicin preparations will be purified from their immunity proteins and tested for their effects in vitro on right side out and inverted membranes. Right side out membranes will be prepared following the methods of Kaback. Inverted membranes will be prepared, as utilized in these studies, from French press disrupted cells.